Following the common cold, otitis media (OM), or inflammation of the middle ear, is the most frequent illness resulting in visits to physicians and the most common cause of hearing impairment and dizziness in children. The recent pathogenic shift of OM by S. pneumoniae vaccination emphasizes the clinical importance of nontypeable Haemophilus influenzae (NTHi), which is the focus of this proposal. In recent years, an alarming increase in antibiotic resistance has been observed worldwide among pathogens. Thus, there is an urgent need to develop new and innovative non-antibiotic approaches to prevent and manage OM. To this end, it is imperative to understand the molecular mechanisms that control the expression of antimicrobial innate immune molecules (AIIMs), which comprise the tubotympanum's first line of defense and determine if these mechanisms can be exploited to prevent or manage OM. Of the AIIMs tested to date, 2-defensin 2 (DEFB4) is the most effective antimicrobial molecule against OM pathogens. However, current understanding of the molecular mechanisms involved in the regulation of the 2-defensin 2 expression by NTHi is limited. The long-term goal of this project is to elucidate the role of innate immunity in OM pathogenesis, focusing on 2-defensin 2, a potent and inducible AIIM and to develop AIIMs-based approaches to prevent or treat OM. Our overall hypotheses are: a) NOD2- and TLR2-mediated activation of NF-:B is required for NTHi- induced 2-defensin 2 up-regulation and contributes to NTHi clearance from the middle ear; b) NTHi-induced IL- 10 negatively regulates NTHi-induced 2-defensin 2 up-regulation and impairs NTHi clearance from the middle ear; and c) Exogenous and over-expressed 2-defensin 2 may have therapeutic potential in the prevention and the management of OM. To evaluate our hypotheses, we will: 1) identify the signaling network(s) involved in NTHi-induced 2-defensin 2 up-regulations (Specific Aim 1); 2) determine the role of IL-10 as a negative regulator of NTHi-induced 2-defensin 2 expression (Specific Aim 2); and 3) determine the therapeutic potential of 2-defensin 2 in the prevention and the management of OM (Specific Aim 3). The significance of this study is: 1) to provide insight into understanding how antimicrobial innate immune molecules (AIIMs) produced by the host protect the middle ear from invading pathogens, and 2) to facilitate the application of AIIMs, particularly 2-defensin 2, in the prevention of OM (using a gene-based approach), and the management of OM (using synthetic AIIMs). Otitis media (OM), or middle ear infection, is one of the most frequent illnesses affecting young children and the most frequent reason for antibiotic prescriptions. The rapid increase in antibiotic resistance among OM pathogens has become a major global health concern. The goal of this proposal is to understand the interaction between bacteria and host and to develop innovative new approaches in preventing and managing OM, without antibiotics, using natural antimicrobial molecules produced by the host.